Cultivation and Genetic Manipulation of Free-Living and Pathogenic Leptospires

钩端螺旋体病是由钩端螺旋体属成员引起的全球性人畜共患疾病。虽然钩端螺旋体病广泛传播，有时甚至致命，但它被认为是一种被忽视和研究不足的疾病。钩端螺旋体病的病原体于1916年首次被鉴定，但体外生长速度慢和操纵该螺旋体基因组的遗传工具有限阻碍了毒力因子的鉴定和疫苗的开发。 钩端螺旋体可大致分为两类：自由生活的寄生虫和感染性病原体。最广泛使用和研究的物种是L. biflexa和L.问号（病原体）。而非致病性L. biflexa比致病L.更容易培养，更适合遗传操作。质问者因此，我们把重点放在L。biflexa掌握操纵该属所需的微生物和遗传技术，目的是将这种专门知识转移到更难治的致病菌株。靶向基因失活、穿梭载体转化和转座子诱变都已成功地应用于L. biflexa。迄今为止，还没有针对致病菌的穿梭载体系统，也很少有关于L.质问者转座子诱变可应用于L.但是它的功能效率很低，以致于不能用于任何广泛的应用，例如营养缺陷型筛选或标记突变。由于缺乏穿梭载体，L.问号阻碍互补作用，因此限制了转座子或靶向缺失突变体的任何所得表型的解释。自L. biflexa比其他物种有更好的转化频率，我们计划优化这种生物的新技术。 于二零一四财政年度，我们已开始评估可能影响钩端螺旋体转化效率的不同系统。λ红重组酶系统已成功地用于其他细菌以改善靶向诱变。我们已经开始在L.如果它看起来有希望，我们将在病原体L中测试它。质问者此外，我们正在研究存在于L.而L. biflexa。该系统的目标和降解外源DNA，我们假设，它可能有助于较低的转化频率在病原体中观察到的相对于腐殖酸。具体来说，我们已经证明CRISPR/cas操纵子在体外生长期间被转录，并且已经将部分操纵子整合到L. biflexa，并且已经表明这些基因也在这种异源宿主中转录。目前，我们正试图在L.并将整个操纵子移入L. biflexa。 我们在2014财年着手开发体外培养L. biflexa来鉴定来自膜级分和可溶级分的高表达蛋白。我们已经确定了丰富表达的蛋白质，可用作细胞标记物，作为基因表达研究的对照，也定量了这些基因的一个子集的转录数据。此外，我们证明了大量的L。Biflexa蛋白进行翻译后修饰，包括磷酸化和乙酰化。高表达的蛋白质使我们能够识别可能发挥重要生理作用的靶点，并用作各种表达研究的标记蛋白。这项工作正在与NIAID持续性病毒性疾病实验室的James卡罗尔博士和NIAID研究技术分支的丽莎奥拉诺博士进行内部合作。 本项目的长期目标是利用改进的工具和技术来了解钩端螺旋体的基本生理和钩端螺旋体的感染和致病机制。质问者这些知识应有助于加速制定预防钩端螺旋体病的措施。

Leptospirosis is a global, zoonotic disease caused by members of the genus Leptospira. Although widespread and sometimes fatal, leptospirosis is considered a neglected and understudied disease. The causative agent of Leptospirosis was first identified in 1916 but the slow in vitro growth rate and limited genetic tools with which to manipulate the genome of this spirochete have hampered the identification of virulence factors and development of a vaccine. Leptospires can be broadly divided into two groups: free-living saprophytes and infectious pathogens. The most widely used and studied species are L. biflexa (a non-pathogenic saprophyte) and L. interrogans (a pathogen). However, the non-pathogenic L. biflexa is more easily cultivated and more amenable to genetic manipulation than the pathogenic L. interrogans. Therefore, we have focused on L. biflexa to master the microbial and genetic techniques needed to manipulate this genus, with the intention to transfer this expertise to the more refractory pathogenic strains. Targeted gene inactivation, shuttle vector transformation, and transposon mutagenesis have all been successfully used in L. biflexa. To date, no shuttle vector system exists for pathogenic species and there are few published reports of targeted gene inactivation in L. interrogans. Transposon mutagenesis can be applied to L. interrogans but it functions at such a low efficiency that it cannot be utilized for any broad applications, such as auxotrophic screens or signature tagged mutagenesis. The lack of a shuttle vector for L. interrogans hinders complementation and thus limits interpretation of any resulting phenotypes of transposon or targeted deletion mutants. Since L. biflexa has a better transformation frequency than other species we plan to optimize new techniques in this organism. In FY2014 we have begun to evaluate different systems that may affect the transformation effiencies of leptospires. The lamda red recombinase system has been used successfully in other bacteria to improve targeted mutagenesis. We have begun to assess this system in L. biflexa, and if it appears promising, we will test it in the pathogen L. interrogans. Also, we are studying the CRISPR/Cas system that is present in L. interrogans but absent in L. biflexa. This system targets and degrades foreign DNA and we hypothesize that it may contribute to the lower transformation frequency observed in the pathogen relative to the saprophyte. Specifically, we have demonstrated that the CRISPR/cas operon is transcribed during in vitro growth and have integrated part of the operon into L. biflexa and have shown that the genes are also transcribed in this heterologous host. Currently, we are attempting to inactivate specific cas genes in L. interrogans and move the entire operon into L. biflexa. We proceeded in FY2014 to develop a proteomic map of in vitro cultivated L. biflexa to identify highly expressed proteins from membrane- and soluble-fractions. We have identified abundantly-expressed proteins that can be used as cellular markers, as controls for gene expression studies, and also quantified the transcript data from a subset of these genes. Further, we demonstrated that a significant number of L. biflexa proteins are subject to post-translational modification including phosphorylation and acetylation. Highly expressed proteins allow us to identify targets that may play important physiological roles and also use as tagged proteins for various expression studies. This work is being completed with an internal collaboration with Dr. James Carroll in the Laboratory of Persistent Viral Diseases, NIAID and Dr. Lisa Olano of the Research Technologies Branch, NIAID. The long-term objective of this project is to use the improved tools and techniques to understand the basic physiology of leptospires and the mechanisms of infection and pathogenecity of L. interrogans. Together this knowledge should help accelerate the development of preventative measures against Leptospirosis.